It has been widely known that human epidermal growth factor receptor 2 (HER2) inhibitors exhibit distinct antitumor responses against HER2-positive breast cancer. To date, Lapatinib (Tykerb ®) has been approved by the U.S. Food and Drug Administration (FDA) as a reversible HER2 inhibitor for treating breast cancer. However, HER2 L755S, T798I and T798M mutations confer drug resistance to lapatinib, restricting its efficacy toward HER2-positive breast cancer. Thus, novel therapy toward mutant HER2 is highly desired. Although several irreversible HER2 inhibitors have been developed to overcome these drug resistance problems, most of them were reported to cause severe side effects. In this study, three pharmacophore models based on HER2 L755S, T798I and T798M mutant structures were constructed and then validated through receiver operating characteristic (ROC) curve analysis and Güner-Henry (GH) scoring methods. Subsequently, these well-validated models were utilized as 3D queries to identify novel irreversible HER2 inhibitors from National Cancer Institute (NCI) database. Finally, two potential irreversible HER2 inhibitor candidates, NSC278329 and NSC718305, were identified and validated through molecular docking, molecular dynamics (MD) simulations and ADMET prediction. Furthermore, the analyses of binding modes showed that both NSC278329 and NSC718305 exhibit good binding interactions with HER2 L755S, T798I and T798M mutants. All together, the above results suggest that both NSC278329 and NSC718305 can serve as novel and effective irreversible HER2 inhibitors for treating breast cancers with HER2 L755S, T798I and T798M mutants. In addition, they may act as lead compounds for designing new irreversible HER2 inhibitors Open Access *Equal contribution.